JP4336400B2 - Enzyme inducer and enzyme production method using the same - Google Patents

Description

（Ｒは炭素数１〜１０のアルキル基、アラルキル基又はアリール基を示す。）本発明誘導剤において、Ｒは、好ましくは、炭素数１〜４のアルキル基、ベンジル基又は置換基を有していてもよいフェニル基もしくはウンベリフェリル基であり、これらの中でも炭素数１〜４のアルキル基、又は置換基を有していてもよいフェニル基もしくはウンベリフェリル基が好ましい。さらに好ましくは、Ｒは、炭素数１〜２のアルキル基、ニトロフェニル基又はメチルウンベリフェリル基である。
【００１０】
本発明誘導剤は、好ましくは、さらにグルコースを有効成分として含有する。
【００１１】
また、本発明は、本発明誘導剤を含む培地において、ＫＤＮａｓｅ生産能を有する微生物をＫＤＮａｓｅの生産条件下において培養する工程、および、培養により得られた培養物からＫＤＮａｓｅを採取する工程を少なくとも含む、ＫＤＮａｓｅの製造方法（以下、本発明製造方法という）を提供する。
【００１２】
本発明製造方法は、好ましくは、下記の工程を少なくとも含む。
（工程１）本発明誘導剤を、ＫＤＮａｓｅ生産能を有する微生物を含有する培養物に添加する。
（工程２）工程１で得られた培養物をＫＤＮａｓｅの生産条件下において培養する。
（工程３）工程２で得られた培養物からＫＤＮａｓｅを採取する。
【００１３】
本発明製造方法において、ＫＤＮａｓｅ生産能を有する微生物は、好ましくは、スフィンゴバクテリウム属に属する細菌である。スフィンゴバクテリウム属に属する細菌は、好ましくは、スフィンゴバクテリウム ｍＯＬ１２−４ｓ（ＦＥＲＭ ＢＰ−５１１６）である。
【００１４】
【発明の実施の形態】
以下に、本発明の実施の形態について説明する。
＜１＞本発明誘導剤
本発明誘導剤は、下記一般式(1)で示されるＫＤＮ誘導体を有効成分として含有する、ＫＤＮａｓｅの誘導剤である。
【００１５】
【化３】

（Ｒは炭素数１〜１０のアルキル基、アラルキル基又はアリール基を示す。）
なお上記一般式(1)におけるＲは、炭素数１〜１０のアルキル基（具体的には、メチル基、エチル基、プロピル基、ブチル基、ペンチル基、ヘキシル基、ヘプチル、オクチル基、ノニル基又はデシル基）、アラルキル基又はアリール基である限りにおいて特に限定されないが、以下に好ましいＲを例示する。
【００１６】
上記一般式(1)におけるＲが炭素数１〜１０のアルキル基であるもののなかでも、Ｒが炭素数１〜４のアルキル基（具体的にはメチル基、エチル基、プロピル基又はブチル基）であるものが好ましく、Ｒが炭素数１〜２のアルキル基（具体的にはメチル基又はエチル基）であるものがより好ましく、Ｒがエチル基であるものが特に好ましい。
【００１７】
上記一般式(1)におけるＲがアラルキル基であるもののなかでも、Ｒがベンジル基であるものが好ましい。
【００１８】
上記一般式(1)におけるＲがアリール基であるもののなかでも、Ｒが置換基を有していてもよいフェニル基もしくはウンベリフェリル基であるものが好ましい。置換基としては、炭素数１〜４のアルキル基、ニトロ基などが挙げられ、置換基は複数であってもよく、複数の場合は互いに同一でも相違してもよい。Ｒがニトロフェニル基又はメチルウンベリフェリル基であるものがより好ましい。
【００１９】
上記一般式(1)におけるＲがニトロフェニル基であるもののなかでも、Ｒがｐ−ニトロフェニル基であるものがより好ましい。
【００２０】
上記一般式(1)におけるＲがメチルウンベリフェリル基であるもののなかでも、Ｒが４−メチルウンベリフェリル基であるものがより好ましい。
【００２１】
なお、上記ＫＤＮ誘導体には、一般式(1)中のカルボン酸基が塩になっているものも包含される。
【００２２】
これらのＫＤＮ誘導体は、ＫＤＮと前記の「Ｒ」に該当する基とを、常法によりグリコシド結合させることにより製造することができる。具体的なＫＤＮ誘導体の製造法については、後述の実施例において詳述する。
【００２３】
本発明誘導剤は、上記一般式(1)で示されるＫＤＮ誘導体を有効成分として含有するが、さらにグルコースを有効成分として含有することが好ましい。すなわち本発明誘導剤は、上記一般式(1)で示されるＫＤＮ誘導体及びグルコースを有効成分として含有することが好ましい。
【００２４】
ここで、上記一般式(1)で示されるＫＤＮ誘導体及びグルコースを有効成分として含有する本発明誘導剤の提供形態は、必ずしも当該ＫＤＮ誘導体とグルコースとの混合物である必要はなく、例えば下記のような提供形態が例示される。
【００２５】
(A)上記一般式(1)で示されるＫＤＮ誘導体とグルコースとの混合物を調製し、これを本発明誘導剤として提供する形態。
【００２６】
(B)上記一般式(1)で示されるＫＤＮ誘導体とグルコースとを別々の容器に封入し、これらをセットで本発明誘導剤として提供する形態。
【００２７】
(C)上記一般式(1)で示されるＫＤＮ誘導体又はグルコースのみを、その使用の際にグルコース又は上記一般式(1)で示されるＫＤＮ誘導体を添加せしめる旨の表示をして、本発明誘導剤として提供する形態。
【００２８】
なお、上記一般式(1)で示されるＫＤＮ誘導体とグルコースとの混合物として本発明誘導剤を提供する場合の混合比は特に限定されないが、ＫＤＮ誘導体：グルコース＝１：１程度が好ましい。
【００２９】
本発明誘導剤は、上記ＫＤＮ誘導体及びグルコースの他、培養において許容可能な担体や添加剤を含んでいてもよい。
【００３０】
なお、ＫＤＮａｓｅ誘導の際に用いる本発明誘導剤の使用量については、後述の「＜２＞本発明製造方法」において詳述する。
【００３１】
＜２＞本発明製造方法
本発明製造方法は、上記の本発明誘導剤を含有する培地で、ＫＤＮａｓｅ生産能を有する微生物をＫＤＮａｓｅの生産条件下において培養する工程、及び、培養により得られた培養物からＫＤＮａｓｅを採取する工程を少なくとも含む、ＫＤＮａｓｅの製造方法である。
【００３２】
ＫＤＮａｓｅ生産能を有する微生物のＫＤＮａｓｅ生産条件下での培養は、この微生物を本発明誘導剤を含まない培地で培養後、培養物に本発明誘導剤を添加して行ってもよいし、培養物を本発明誘導剤を含む培地に接種して行ってもよい。
【００３３】
本発明製造方法は、下記の工程を少なくとも含むことが好ましい。
（工程１）本発明誘導剤を、ＫＤＮａｓｅ生産能を有する微生物を含有する培養物に添加する。
（工程２）工程１で得られた培養物をＫＤＮａｓｅの生産条件下において培養する。
（工程３）工程２で得られた培養物からＫＤＮａｓｅを採取する。
【００３４】
ここで、ＫＤＮａｓｅ生産能を有する微生物は特に限定されないが、スフィンゴバクテリウム属に属する細菌であることが好ましく、スフィンゴバクテリウムｍＯＬ１２−４ｓ（ＦＥＲＭ ＢＰ−５１１６）であることがより好ましい。
【００３５】
このスフィンゴバクテリウム ｍＯＬ１２−４ｓ（ＦＥＲＭ ＢＰ−５１１６）は、下記の酵素学的性質を有するＫＤＮａｓｅを生産する。
▲１▼作用：
ＫＤＮを含有する複合糖質もしくは糖質に作用し、ＫＤＮケトシド結合を加水分解して、ＫＤＮを含有しない複合糖質もしくは糖質又はＫＤＮが部分的に除去された複合糖質もしくは糖質と、遊離のＫＤＮとを生成する。
▲２▼基質特異性：
ＫＤＮを含有する複合糖質もしくは糖質には作用するが、Ｎ−アセチルノイラミン酸又はＮ−グリコリルノイラミン酸を含有する複合糖質もしくは糖質における、Ｎ−アセチルノイラミン酸又はＮ−グリコリルノイラミン酸のケトシド結合には作用しない。
【００３６】
このＫＤＮａｓｅは、さらに下記の性質を有する。
(i)至適反応ｐＨ：
ｐＨ６付近
(ii)安定ｐＨ範囲
２５℃においてｐＨ４〜９で安定
(iii)至適反応温度
２５℃付近
(iv)熱安定性
２５℃で少なくとも４８時間失活しない。
(v)阻害及び安定化
遊離のＫＤＮによって阻害される。ウシ血清アルブミン存在下で安定化される。
【００３７】
従って、本発明製造方法によって製造されるＫＤＮａｓｅは、上記の酵素学的性質を有するものであることが好ましい。
【００３８】
ＫＤＮａｓｅ生産条件とは、ＫＤＮａｓｅ生産能を有する微生物がＫＤＮａｓｅを生産する条件を意味し、使用する微生物によって適宜選択されるが、通常には、この微生物が資化可能な炭素源（グルコースなど）、窒素源（酵母エキス、ペプトン、肉エキス、コーンスティープリカー、大豆粕、カゼインの加水分解物(casamino acids)などの有機窒素源、塩酸アンモニウム、硫酸アンモニウム、尿素、硝酸アンモニウムなどの無機窒素源、無機塩（カルシウム、マグネシウム、カリウム、ナトリウムなどの硫酸塩、リン酸塩、塩酸塩など）などを含む培地中で、好気的な培養法（振盪培養、通気攪拌培養など）によって生育に適した温度で数時間〜数日間培養するという条件が挙げられる。
【００３９】
ＫＤＮａｓｅ生産条件下での培養における本発明誘導剤の使用量は、本発明誘導剤を使用しないときに比べ、産生されるＫＤＮａｓｅの量が増大するのに十分な量であればよく、通常には、培地中のＫＤＮ誘導体の濃度として０．０１〜１％(w/v)、好ましくは０．０１〜０．３％(w/v)である。
【００４０】
ＫＤＮａｓｅ生産条件下での培養における培地中のグルコースの量は、ＫＤＮａｓｅの誘導を増大させるのに十分な量であればよく、通常には、培地中の濃度で０．０１〜１％(w/v)、好ましくは０．０１〜０．３％(w/v)である。従って、本発明誘導剤に任意成分として含まれるグルコースは、培地中に十分量のグルコースが存在する場合には、なくてもよい。
【００４１】
その他、ＫＤＮａｓｅ生産条件下での培養における、ＫＤＮａｓｅ誘導のための、初期細胞密度、培養時間等の条件は、後記実施例２に示すように、通常に行われる実験手順によって設定することができる。
【００４２】
培養により得られた培養物からのＫＤＮａｓｅの採取は、ＷＯ９６／００７８１号公報に記載されたＫＤＮａｓｅの取得法に従って行うことができる。例えば、培養物から遠心分離などで収集した菌体から、超音波処理などによる破砕、あるいは浸透圧ショック等の適当な方法によって放出された酵素を含む画分を得、次いで、例えば、硫酸アンモニウム（硫安）、硫酸ナトリウム等による塩析、透析、限外濾過法、吸着クロマトグラフィー、陰イオン及び陽イオン交換クロマトグラフィー、疎水性クロマトグラフィー、ゲル濾過法、電気泳動法などの公知の酵素精製法、さらにはＫＤＮ含有糖タンパク質（KDN-gp）を結合させたアガロースゲル等を用いたアフィニティクロマトグラフィーによって目的とする精製度の酵素標品を得ることができる。
【００４３】
【実施例】
以下に、本発明を実施例により具体的に説明する。しかしながら、これらにより本発明の技術的範囲が限定されるべきものではない。
【００４４】
【調製例１】
ＫＤＮ誘導体等の調製
ＫＤＮオリゴ糖アルジトール画分（ＫＤＮ−ＯＳ）は、ニジマス卵巣液から、既報（J. Biol. Chem. 271, 2909-2913(1996)）の方法に従って調製した。ＫＤＮ、ｐ−α−ニトロフェニルＫＤＮ（ＫＤＮα２ｐＮＰ）及びα−４−メチルウンベリフェリルＫＤＮ（ＫＤＮα２ＭｅＵｍｂ）は、既報（Chem. Pharm. Bull. 37, 821-823(1989); Chem. Ber. 101, 1089-1094(1968); Chem. Pharm. Bull. 43, 1844-1848(1995)）の方法に従って合成した。α−メチルＫＤＮ（ＫＤＮα２Ｍｅ）、α−エチルＫＤＮ（ＫＤＮα２Ｅｔ）及びα−ベンジルＫＤＮ（ＫＤＮα２Ｂｎ）は、以下の様にして調製した（図１）。メチル 4,5,7,8,9-ペンタ-O-アセチル-2-クロロ-2,3-ジデオキシ-D-glycero-β-D-galacto-2-ノヌロピラノソネート(2, 1.0 g, 1.96 mmol) を、メタノール、エタノール及びベンジルアルコール(30 ml)にそれぞれ溶解し、攪拌しながら安息香酸ナトリウム(0.34 g, 2.36 mmol) を加えた。得られた懸濁液を室温で０．５〜３時間攪拌し、反応液をセライト(Celite)で濾過し、濾液をエバポレーターにより減圧乾固した。残渣を酢酸エチル(30 ml)に溶解し、この溶液を５％炭酸水素ナトリウム水溶液で２回洗浄し、ブラインで洗浄し、硫酸ナトリウム上で乾燥した後、エバポレーターにより減圧乾固した。残渣を、ｎ−ヘキサン／酢酸エチル(2:1, v/v)を用いてシリカゲル(Merck, Kieselgel 60, 70-230 mesh, ASTM, 80 g)カラムのクロマトグラフィーに付し、無色のアモルファス粉末として、3, 4及び5の化合物を得た。各生成物を、ｎーヘキサン／酢酸エチルから結晶化した。脱保護のために、各結晶をメタノール(10 ml)に溶解し、１Ｎ水酸化ナトリウム(10 ml)を加えた。得られた溶液を室温で４時間攪拌し、次いで水で希釈し、Dowex 50W-X8(H⁺)樹脂を用いて０℃で脱イオンした。濾液を凍結乾燥し、6, 7及び8の化合物を無色の粉末として得た。
【００４５】
得られた化合物の性質を以下の通り確認した。
【００４６】
メチル 2-デオキシ-D-glycero-α-D-galacto-2-ノヌロピラノシドン酸(6, KDNα2Me): 収率 98%. [α]_D ²⁵ -29.2° (c = 1.0, CH₃OH).元素分析 C₁₀H₁₈O₉の計算値: C, 42.55; H, 6.43.測定値: 42.44; H, 6.48. IRデータ, ν_max ^film cm^-1: 1720 (C=O). ¹H-NMR (300 MHz, D₂O) data:δ= 1.57 (1H, t, J = 12.0 Hz, 3-H_ax), 2.64 (1H, dd, J = 5.0, 12.0 Hz, 3-H_eq), 3.31 (3H, s, 2-OCH₃), 3.49 (1H, dd, J = 9.0, 9.5 Hz, 5-H), 3.57 (1H, ddd, J = 5.0, 9.0, 12.0 Hz, 4-H), 3.65 (1H, dd, J = 2.0, 9.5 Hz, 6-H), 3.66 (1H, dd, J = 6.5, 12.5 Hz, 9-H), 3.85 (1H, dd, J = 2.0, 8.5 Hz, 7-H), 3.88 (1H, ddd, J = 2.5, 6.5, 8.5 Hz, 8-H), 3.88 (1H, dd, J = 2.5, 12.5 Hz, 9'-H).
エチル 2-デオキシ-D-glycero-α-D-galacto-2-ノヌロピラノシドン酸(7, KDNα2Et): 収率 98%. [α]_D ²⁵ -34.2°(c = 1.0, CH₃OH).元素分析 C₁₁H₂₀O₉の計算値: C, 44.59; H, 6.80.測定値: 44.64; H, 6.58. IRデータ, ν_max ^film cm^-1: 1720 (C=O). ¹H-NMR (300 MHz, D₂O): δ= 1.14 (3H, t, J = 7.0 Hz, -CH₂CH₃), 1.57 (1H, dd, J = 11.5, 12.0 Hz, 3-H_ax), 2.66 (1H, dd, J = 4.5, 12.0 Hz, 3-H_eq), 3.46 (1H, dq, J = 9.0, 7.0 Hz, 2-OCHCH₃), 3.48 (1H, t, J = 9.0 Hz, 5-H), 3.56 (1H, ddd, J = 4.5, 9.0, 11.5 Hz, 4-H), 3.65 (1H, dd, J = 2.0, 9.0 Hz, 6-H), 3.66 (1H, dd, J = 3.0, 13.0 Hz, 9-H), 3.76 (1H, dq, J = 9.0, 7.0 Hz, 2-OCHCH₃), 3.83 (1H, ddd, J = 1.5, 3.0, 9.0 Hz, 8-H), 3.85 (1H, dd, J = 2.0, 9.0 Hz, 7-H), 3.88 (1H, dd, J = 1.5, 13.0 Hz, 9'-H).
ベンジル 2-デオキシ-D-glycero-α-D-galacto-2-ノヌロピラノシドン酸(8, KDNα2Bn): 収率 98%. [α]_D ²⁵ -32.0°(c = 1.0, CH₃OH).元素分析 C₁₆H₂₂O₉の計算値: C, 53.63; H, 6.19.測定値: C, 53.64; H, 6.38. IRデータ, ν_max ^film cm^-1: 1720 (C=O), 1600 (フェニル).¹H-NMR (300 MHz, D₂O): δ= 1.64 (1H, t, J = 12.0 Hz, 3-H_ax), 2.70 (1H, dd, J = 4.5, 12.0 Hz, 3-H_eq), 3.56 (1H, dd, J = 9.0, 9.5 Hz, 5-H), 3.61 (1H, ddd, J = 4.5, 9.0, 12.0 Hz, 4-H), 3.69 (1H, dd, J = 6.5, 11.5 Hz, 9-H), 3.70 ( 1H, dd, J = 2.0, 9.5 Hz, 6-H), 3.80 (1H, dd, J = 2.0, 9.0 Hz, 7-H), 3.84 (1H, ddd, J = 2.0, 6.5, 9.0 Hz, 8-H), 3.88 (1H, dd, J = 2.0, 11.5 Hz, 9'-H), 4.49 (1H, d, J = 11.0 Hz, C₆H₅CH-), 4.73 (1H, d, J = 11.0 Hz, C₆H₅CH'-), 7.34-7.45 (5H, C₆H₅-).
【００４７】
【実施例１】
ＫＤＮ誘導体によるＫＤＮａｓｅの誘導
スフィンゴバクテリウム ｍＯＬ１２−４ｓの細菌細胞を、１％(w/v)のカゼイン加水分解物(casamino acids、ギブコ製)及び１％(w/v)のグルコース(Glc)を含むＭ９液体培地中で２５℃で培養した。増殖期の菌体（細胞）を集め、Ｍ９液体培地で２回洗浄した。表１に示す添加物質をそれぞれ０．１％(w/v)の濃度で含む、それぞれ２．０ｍｌのＭ９液体培地に１×１０⁹個の細胞を接種し、２５℃で１２時間インキュベートし、細胞内ＫＤＮａｓｅ活性を測定した。細胞内ＫＤＮａｓｅ活性は、培養液を13,000rpm(14,000×ｇ)で１０分間遠心することにより集めた細胞を、0.1M NaClを含む0.1Mトリス−酢酸緩衝液(pH 6.0)の0.25ｍｌに懸濁し、超音波破砕した（20ワット、30秒間）。これを13,000rpm(14,000×ｇ)で１０分間遠心した後に得られた上清のＫＤＮａｓｅ活性を測定し、２ｍｌの培養液当たりのユニットとして算出した。なお、ＫＤＮａｓｅ活性は、基質として1.4nmolのＫＤＮα２ＭｅＵｍｂを、20μlの上清に加えて、25℃で30分間反応させ、反応後、2.5mlの85mMグリシン炭酸塩緩衝液(pH 9.3)と混合して、蛍光強度を測定した。１分間に１nmolのＫＤＮα２ＭｅＵｍｂを加水分解する酵素量を１ユニットと定義した。結果を表１に示す。
【００４８】
【表１】
表１ 各種物質添加時のＫＤＮａｓｅ活性
────────────────────────────────
添加物質 ＫＤＮａｓｅ活性（ユニット/2 ml培養液）
────────────────────────────────
ＫＤＮα２Ｍｅ＋Ｇｌｃ ７．３
ＫＤＮα２Ｅｔ＋Ｇｌｃ １１．０
ＫＤＮα２Ｂｎ＋Ｇｌｃ ５．１
ＫＤＮα２ｐＮＰ＋Ｇｌｃ ９．０
ＫＤＮα２ＭｅＵｍｂ＋Ｇｌｃ ９．４
ＫＤＮ−ＯＳ＋Ｇｌｃ ２．１
ＫＤＮ＋Ｇｌｃ ０．３
Ｇｌｃ ０．７
ＫＤＮα２Ｂｎ ０．２
ＫＤＮ−ＯＳ ４．５
ＫＤＮ ０．１
────────────────────────────────
注）ＫＤＮα２Ｍｅ、ＫＤＮα２Ｅｔ、ＫＤＮα２ｐＮＰ及びＫＤＮα２ＭｅＵｍｂの単独の添加時の活性は、０．３〜１．８であり、ＫＤＮα２Ｂｎとほぼ同じであった。
【００４９】
表１から、グルコースが存在するときに、一般式(1)で示されるＫＤＮ誘導体は、顕著なＫＤＮａｓｅ誘導活性を示すことが明らかである。また、グルコースは０．１％（w/v)の量が存在するだけで一般式(1)で示されるＫＤＮ誘導体のＫＤＮａｓｅ誘導活性が発揮されるので、一般の培養条件で炭素源として加えられるレベルのグルコースが存在する条件では、一般式(1)で示されるＫＤＮ誘導体のみをＫＤＮａｓｅ誘導剤として加えることで十分なことが分かる。さらに、このような条件では、ＫＤＮ−ＯＳに比べ一般式(1)で示されるＫＤＮ誘導体のＫＤＮａｓｅ誘導活性が顕著に高いことが分かる。
【００５０】
【実施例２】
ＫＤＮａｓｅ誘導条件の最適化
誘導剤としてＫＤＮα２ＭｅＵｍｂを用いた場合の、スフィンゴバクテリウムｍＯＬ１２−４ｓのＫＤＮａｓｅの誘導条件を検討した。
【００５１】
(a) 初期細胞密度
１％(w/v)のカゼイン加水分解物(casamino acids、ギブコ製)及び１％(w/v)のグルコース(Glc)を含むＭ９液体培地中で２５℃で培養した細胞を、種々の細胞密度で、０．１％(w/v)ＫＤＮα２ＭｅＵｍｂ及び０．１％(w/v)Ｇｌｃを含むＭ９液体培地に接種し、２５℃で１２時間インキュベートし、実施例１と同様の方法で細胞内ＫＤＮａｓｅ活性を測定した。結果を図２の(a)に示す。誘導の至適初期細胞密度は１．０×１０⁹細胞／ｍｌであった。
【００５２】
(b) 誘導剤濃度
１％(w/v)のカゼイン加水分解物(casamino acids、ギブコ製)及び１％(w/v)のグルコース(Glc)を含むＭ９液体培地中で２５℃で培養した細胞を、種々の濃度のＫＤＮα２ＭｅＵｍｂ及び０．１％(w/v)Ｇｌｃを含むＭ９液体培地に、１．０×１０⁹細胞／ｍｌの細胞密度で接種し、２５℃で１２時間インキュベートし、実施例１と同様の方法で細胞内ＫＤＮａｓｅ活性を測定した。結果を図２の(b)に示す。０．０１％(w/v)の濃度でＫＤＮａｓｅの誘導に効果的であった。
(c) 誘導時間
１％(w/v)のカゼイン加水分解物(casamino acids、ギブコ製)及び１％(w/v)のグルコース(Glc)を含むＭ９液体培地中で２５℃で培養した細胞を、０．１％(w/v)ＫＤＮα２ＭｅＵｍｂ及び０．１％(w/v)Ｇｌｃを含むＭ９液体培地に、１．０×１０⁹細胞／ｍｌの細胞密度で接種し、２５℃で種々の時間インキュベートし、実施例１と同様の方法で細胞内ＫＤＮａｓｅ活性を測定した。結果を図２の(c)に示す。６時間〜２４時間で効果的なＫＤＮａｓｅの誘導が得られた。
【００５３】
【発明の効果】
本発明によれば、ＫＤＮａｓｅ誘導作用の高いＫＤＮａｓｅの誘導剤及びそれを用いたＫＤＮａｓｅの製造方法が提供され、ＫＤＮａｓｅを効率よく製造することができる。なお、本発明誘導剤の有効成分であるＫＤＮ誘導体は、調製が容易であり、安価かつ大量に提供することができる。
【図面の簡単な説明】
【図１】 ＫＤＮ誘導体の合成スキームを示す。
【図２】 ＫＤＮａｓｅ誘導条件の一例を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an enzyme inducer and an enzyme production method. In more detail, it is related with the inducer and manufacturing method of a deamino neuraminidase.
[0002]
[Prior art]
Deaminoneuraminic acid (3-deoxy-D-glycero-D-galacto-nonulosonic acid or 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid; hereinafter also referred to as KDN) is a complex It is widely known as a constituent of carbohydrates in the biological world, and is known to have various modes of existence, and an enzyme that cleaves KDN ketoside bonds in carbohydrates containing KDN (deaminoneuraminidase; hereinafter referred to as KDNase). Also known as). KDNase is expected as a useful reagent for analyzing the structure and function of KDN and detecting KDN.
[0003]
WO96 / 00781 discloses KDNase, a production method thereof, and a KDNase-producing bacterium (Sphingobacterium mOL12-4s (FERM BP-5116)). In the publication, sphingobacterium mOL12-4s (FERM BP-5116) is contained in a medium containing KDN oligosaccharide alcohol (KDNα2 → 3Galβ1 → 3GalNAcα1 → 3 [KDNα2 → (8KDNα2 →) _n → 6] GalNAcol). It is described that KDNase can be induced by culturing.
[0004]
However, a KDNase inducer containing KDN glycoside as an active ingredient and a method for producing KDNase using the same are not disclosed.
[0005]
[Problems to be solved by the invention]
If it is possible to provide an inducer having a KDNase-inducing action that exceeds that of KDN oligosaccharide alcohol and a method for producing KDNase using the same, KDNase can be produced extremely efficiently and in large quantities using KDNase-producing bacteria, and KDNase can be provided at a lower cost. can do.
[0006]
Therefore, the problem to be solved by the present invention is to provide a novel KDNase inducer and a method for producing KDNase using the same.
[0007]
[Means for Solving the Problems]
As a result of intensive studies to solve the above-mentioned problems, the present inventor has found that a specific KDN glycoside has a remarkable KDNase-inducing action exceeding that of the KDN oligosaccharide alcohol, and has completed the present invention. .
[0008]
That is, the present invention provides a KDNase inducer (hereinafter referred to as the present invention inducer) containing a KDN derivative represented by the following general formula (1) as an active ingredient.
[0009]
[Chemical formula 2]

(R represents an alkyl group having 1 to 10 carbon atoms, an aralkyl group or an aryl group.) In the inducer of the present invention, R preferably has an alkyl group having 1 to 4 carbon atoms, a benzyl group or a substituent. It may be a phenyl group or umbelliferyl group, and among them, an alkyl group having 1 to 4 carbon atoms or a phenyl group or umbelliferyl group which may have a substituent is preferable. More preferably, R is an alkyl group having 1 to 2 carbon atoms, a nitrophenyl group or a methylumbelliferyl group.
[0010]
The inducing agent of the present invention preferably further contains glucose as an active ingredient.
[0011]
Further, the present invention includes at least a step of culturing a microorganism having KDNase-producing ability under a KDNase production condition in a medium containing the inducer of the present invention, and a step of collecting KDNase from the culture obtained by the culture. A method for producing KDNase (hereinafter referred to as the production method of the present invention) is provided.
[0012]
The production method of the present invention preferably includes at least the following steps.
(Step 1) The inducer of the present invention is added to a culture containing a microorganism having the ability to produce KDNase.
(Step 2) The culture obtained in Step 1 is cultured under KDNase production conditions.
(Step 3) KDNase is collected from the culture obtained in Step 2.
[0013]
In the production method of the present invention, the microorganism having the ability to produce KDNase is preferably a bacterium belonging to the genus Sphingobacterium. The bacterium belonging to the genus Sphingobacterium is preferably sphingobacterium mOL12-4s (FERM BP-5116).
[0014]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will be described below.
<1> Inducing Agent of the Present Invention The inducing agent of the present invention is a KDNase inducer containing a KDN derivative represented by the following general formula (1) as an active ingredient.
[0015]
[Chemical 3]

(R represents an alkyl group having 1 to 10 carbon atoms, an aralkyl group, or an aryl group.)
R in the general formula (1) is an alkyl group having 1 to 10 carbon atoms (specifically, methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, heptyl, octyl group, nonyl group). Or a decyl group), an aralkyl group, or an aryl group.
[0016]
Among those in which R in the general formula (1) is an alkyl group having 1 to 10 carbon atoms, R is an alkyl group having 1 to 4 carbon atoms (specifically, a methyl group, an ethyl group, a propyl group, or a butyl group). Are preferable, those in which R is an alkyl group having 1 to 2 carbon atoms (specifically, a methyl group or an ethyl group) are more preferable, and those in which R is an ethyl group are particularly preferable.
[0017]
Among those in which R in the general formula (1) is an aralkyl group, those in which R is a benzyl group are preferable.
[0018]
Among those in which R in the general formula (1) is an aryl group, those in which R is a phenyl group or umbelliferyl group which may have a substituent are preferable. Examples of the substituent include an alkyl group having 1 to 4 carbon atoms and a nitro group. The substituent may be plural, and in the case of plural, they may be the same as or different from each other. More preferably, R is a nitrophenyl group or a methylumbelliferyl group.
[0019]
Among those in which R in the general formula (1) is a nitrophenyl group, those in which R is a p-nitrophenyl group are more preferable.
[0020]
Among those in which R in the general formula (1) is a methylumbelliferyl group, those in which R is a 4-methylumbelliferyl group are more preferable.
[0021]
The KDN derivative includes those in which the carboxylic acid group in the general formula (1) is a salt.
[0022]
These KDN derivatives can be produced by glycoside bonding KDN and a group corresponding to the above “R” by a conventional method. A specific method for producing a KDN derivative will be described in detail in Examples below.
[0023]
The inducer of the present invention contains the KDN derivative represented by the above general formula (1) as an active ingredient, but preferably further contains glucose as an active ingredient. That is, the inducer of the present invention preferably contains a KDN derivative represented by the above general formula (1) and glucose as active ingredients.
[0024]
Here, the form of providing the inducer of the present invention containing the KDN derivative represented by the general formula (1) and glucose as active ingredients is not necessarily a mixture of the KDN derivative and glucose. Various forms of provision are exemplified.
[0025]
(A) A form in which a mixture of a KDN derivative represented by the general formula (1) and glucose is prepared and provided as an inducer of the present invention.
[0026]
(B) A form in which the KDN derivative represented by the general formula (1) and glucose are sealed in separate containers and provided as a set of the inducer of the present invention.
[0027]
(C) Only the KDN derivative or the glucose represented by the above general formula (1) is used, and the indication that the glucose or the KDN derivative represented by the above general formula (1) is added during use thereof is derived from the present invention. Form provided as an agent.
[0028]
The mixing ratio in the case of providing the inducer of the present invention as a mixture of the KDN derivative represented by the general formula (1) and glucose is not particularly limited, but is preferably about KDN derivative: glucose = 1: 1.
[0029]
In addition to the above KDN derivative and glucose, the inducer of the present invention may contain carriers and additives that are acceptable in culture.
[0030]
In addition, the usage-amount of this invention inducer used in the case of KDNase induction | guidance is explained in full detail in "<2> this invention manufacturing method" mentioned later.
[0031]
<2> Production method of the present invention The production method of the present invention comprises a step of culturing a microorganism having a KDNase-producing ability under the KDNase production conditions in a medium containing the above-described inducer of the present invention, and a culture obtained by the culture. A method for producing KDNase, comprising at least a step of collecting KDNase from a product.
[0032]
Cultivation of microorganisms capable of producing KDNase under KDNase production conditions may be performed by culturing the microorganisms in a medium not containing the inducer of the present invention, and then adding the inducer of the present invention to the culture. May be inoculated into a medium containing the inducer of the present invention.
[0033]
The production method of the present invention preferably includes at least the following steps.
(Step 1) The inducer of the present invention is added to a culture containing a microorganism having the ability to produce KDNase.
(Step 2) The culture obtained in Step 1 is cultured under KDNase production conditions.
(Step 3) KDNase is collected from the culture obtained in Step 2.
[0034]
Here, the microorganism having the ability to produce KDNase is not particularly limited, but is preferably a bacterium belonging to the genus Sphingobacteria, and more preferably sphingobacteria mOL12-4s (FERM BP-5116).
[0035]
This sphingobacterium mOL12-4s (FERM BP-5116) produces KDNase having the following enzymatic properties.
(1) Action:
Acting on a glycoconjugate or carbohydrate containing KDN, hydrolyzing the KDN ketoside bond, and a glycoconjugate or carbohydrate containing no KDN, or a glycoconjugate or carbohydrate from which KDN has been partially removed; To produce free KDN.
(2) Substrate specificity:
It acts on complex carbohydrates or carbohydrates containing KDN, but N-acetylneuraminic acid or N- in complex carbohydrates or carbohydrates containing N-acetylneuraminic acid or N-glycolylneuraminic acid It does not affect the ketoside bond of glycolylneuraminic acid.
[0036]
This KDNase further has the following properties.
(i) Optimal reaction pH:
around pH 6
(ii) Stable at pH 4-9 in a stable pH range of 25 ° C
(iii) Optimal reaction temperature around 25 ° C
(iv) Thermal stability No inactivation at 25 ° C. for at least 48 hours.
(v) Inhibited and stabilized by free KDN. Stabilized in the presence of bovine serum albumin.
[0037]
Therefore, the KDNase produced by the production method of the present invention preferably has the above enzymatic properties.
[0038]
KDNase production conditions mean conditions under which a microorganism having KDNase production ability produces KDNase, and is appropriately selected depending on the microorganism to be used. Usually, a carbon source (such as glucose) that can be assimilated by this microorganism, Nitrogen sources (yeast extract, peptone, meat extract, corn steep liquor, soybean meal, organic nitrogen sources such as casein acids, inorganic nitrogen sources such as ammonium hydrochloride, ammonium sulfate, urea, ammonium nitrate, inorganic salts ( In culture medium containing calcium, magnesium, potassium, sodium sulfates, phosphates, hydrochlorides, etc.) at a temperature suitable for growth by aerobic culture methods (shaking culture, aeration and agitation culture, etc.) The condition of culturing for a time to several days is mentioned.
[0039]
The amount of the inducer of the present invention used in the culture under KDNase production conditions may be an amount sufficient to increase the amount of KDNase produced compared to when the inducer of the present invention is not used. The concentration of the KDN derivative in the medium is 0.01 to 1% (w / v), preferably 0.01 to 0.3% (w / v).
[0040]
The amount of glucose in the medium in the culture under KDNase production conditions may be an amount sufficient to increase the induction of KDNase, and is usually 0.01-1% (w / w) at a concentration in the medium. v), preferably 0.01 to 0.3% (w / v). Therefore, the glucose contained as an optional component in the inducer of the present invention may not be present when a sufficient amount of glucose is present in the medium.
[0041]
In addition, conditions such as initial cell density and culture time for KDNase induction in culture under KDNase production conditions can be set by a commonly performed experimental procedure as shown in Example 2 below.
[0042]
Collection of KDNase from the culture obtained by culturing can be performed according to the method for obtaining KDNase described in WO96 / 00781. For example, a fraction containing an enzyme released by an appropriate method such as disruption by sonication or osmotic shock is obtained from bacterial cells collected by centrifugation or the like from the culture, and then, for example, ammonium sulfate (ammonium sulfate) ), Salting out with sodium sulfate, dialysis, ultrafiltration, adsorption chromatography, anion and cation exchange chromatography, hydrophobic chromatography, gel filtration, electrophoresis, and other known enzyme purification methods, Can obtain an enzyme preparation having a desired degree of purification by affinity chromatography using an agarose gel or the like to which KDN-containing glycoprotein (KDN-gp) is bound.
[0043]
【Example】
Hereinafter, the present invention will be specifically described by way of examples. However, the technical scope of the present invention should not be limited by these.
[0044]
[Preparation Example 1]
Preparation of KDN Derivatives, etc. KDN oligosaccharide alditol fraction (KDN-OS) was prepared from rainbow trout ovary according to the method described previously (J. Biol. Chem. 271, 2909-2913 (1996)). KDN, p-α-nitrophenyl KDN (KDNα2pNP) and α-4-methylumbelliferyl KDN (KDNα2MeUmb) have been reported (Chem. Pharm. Bull. 37, 821-823 (1989); Chem. Ber. 101, 1089-1094 (1968); Chem. Pharm. Bull. 43, 1844-1848 (1995)). α-methyl KDN (KDNα2Me), α-ethyl KDN (KDNα2Et) and α-benzyl KDN (KDNα2Bn) were prepared as follows (FIG. 1). Methyl 4,5,7,8,9-penta-O-acetyl-2-chloro-2,3-dideoxy-D-glycero-β-D-galacto-2-nonuropyranosonate (2, 1.0 g, 1.96 mmol) was dissolved in methanol, ethanol and benzyl alcohol (30 ml), respectively, and sodium benzoate (0.34 g, 2.36 mmol) was added with stirring. The obtained suspension was stirred at room temperature for 0.5 to 3 hours, the reaction solution was filtered through Celite, and the filtrate was dried under reduced pressure using an evaporator. The residue was dissolved in ethyl acetate (30 ml), and this solution was washed twice with 5% aqueous sodium hydrogen carbonate solution, washed with brine, dried over sodium sulfate, and then evaporated to dryness with an evaporator. The residue was chromatographed on a silica gel (Merck, Kieselgel 60, 70-230 mesh, ASTM, 80 g) column using n-hexane / ethyl acetate (2: 1, v / v) to give a colorless amorphous powder. As a result, compounds 3, 4, and 5 were obtained. Each product was crystallized from n-hexane / ethyl acetate. For deprotection, each crystal was dissolved in methanol (10 ml) and 1N sodium hydroxide (10 ml) was added. The resulting solution was stirred at room temperature for 4 hours, then diluted with water and deionized at 0 ° C. using Dowex 50W-X8 (H ⁺ ) resin. The filtrate was lyophilized to give 6, 7 and 8 as a colorless powder.
[0045]
The properties of the obtained compound were confirmed as follows.
[0046]
Methyl 2-deoxy-D-glycero-α-D-galacto-2-nonuropyranoside acid (6, KDNα2Me): Yield 98%. [Α] _D ²⁵ -29.2 ° (c = 1.0, CH ₃ OH Elemental analysis Calculated value of C ₁₀ H ₁₈ O ₉ : C, 42.55; H, 6.43.Measured value: 42.44; H, 6.48. IR data, ν _max ^film cm ^-1 : 1720 (C = O). ¹ H -NMR (300 MHz, D ₂ O) data: δ = 1.57 (1H, t, J = 12.0 Hz, 3-H _ax ), 2.64 (1H, dd, J = 5.0, 12.0 Hz, 3-H _eq ), 3.31 (3H, s, 2-OCH ₃ ), 3.49 (1H, dd, J = 9.0, 9.5 Hz, 5-H), 3.57 (1H, ddd, J = 5.0, 9.0, 12.0 Hz, 4-H), 3.65 (1H, dd, J = 2.0, 9.5 Hz, 6-H), 3.66 (1H, dd, J = 6.5, 12.5 Hz, 9-H), 3.85 (1H, dd, J = 2.0, 8.5 Hz, 7 -H), 3.88 (1H, ddd, J = 2.5, 6.5, 8.5 Hz, 8-H), 3.88 (1H, dd, J = 2.5, 12.5 Hz, 9'-H).
Ethyl 2-deoxy-D-glycero-α-D-galacto-2-nonuropyranoside acid (7, KDNα2Et): Yield 98%. [Α] _D ²⁵ -34.2 ° (c = 1.0, CH ₃ OH Elemental analysis Calculated value of C ₁₁ H ₂₀ O ₉ : C, 44.59; H, 6.80.Measured value: 44.64; H, 6.58. IR data, ν _max ^film cm ^-1 : 1720 (C = O). ¹ H -NMR (300 MHz, D ₂ O): δ = 1.14 (3H, t, J = 7.0 Hz, -CH ₂ CH ₃ ), 1.57 (1H, dd, J = 11.5, 12.0 Hz, 3-H _ax ), 2.66 (1H, dd, J = 4.5, 12.0 Hz, 3-H _eq ), 3.46 (1H, dq, J = 9.0, 7.0 Hz, 2-OCHCH ₃ ), 3.48 (1H, t, J = 9.0 Hz, 5 -H), 3.56 (1H, ddd, J = 4.5, 9.0, 11.5 Hz, 4-H), 3.65 (1H, dd, J = 2.0, 9.0 Hz, 6-H), 3.66 (1H, dd, J = 3.0, 13.0 Hz, 9-H), 3.76 (1H, dq, J = 9.0, 7.0 Hz, 2-OCHCH ₃ ), 3.83 (1H, ddd, J = 1.5, 3.0, 9.0 Hz, 8-H), 3.85 (1H, dd, J = 2.0, 9.0 Hz, 7-H), 3.88 (1H, dd, J = 1.5, 13.0 Hz, 9'-H).
Benzyl 2-deoxy-D-glycero-α-D-galacto-2-nonuropyranoside acid (8, KDNα2Bn): Yield 98%. [Α] _D ²⁵ -32.0 ° (c = 1.0, CH ₃ OH Elemental analysis Calculated values for C ₁₆ H ₂₂ O ₉ : C, 53.63; H, 6.19.Measured values: C, 53.64; H, 6.38.IR data, ν _max ^film cm ^-1 : 1720 (C = O), 1600 (phenyl). ¹ H-NMR (300 MHz, D ₂ O): δ = 1.64 (1H, t, J = 12.0 Hz, 3-H _ax ), 2.70 (1H, dd, J = 4.5, 12.0 Hz, 3-H _eq ), 3.56 (1H, dd, J = 9.0, 9.5 Hz, 5-H), 3.61 (1H, ddd, J = 4.5, 9.0, 12.0 Hz, 4-H), 3.69 (1H, dd, J = 6.5, 11.5 Hz, 9-H), 3.70 (1H, dd, J = 2.0, 9.5 Hz, 6-H), 3.80 (1H, dd, J = 2.0, 9.0 Hz, 7-H), 3.84 ( 1H, ddd, J = 2.0, 6.5, 9.0 Hz, 8-H), 3.88 (1H, dd, J = 2.0, 11.5 Hz, 9'-H), 4.49 (1H, d, J = 11.0 Hz, C ₆ H ₅ CH-), 4.73 (1H, d, J = 11.0 Hz, C ₆ H ₅ CH'-), 7.34-7.45 (5H, C ₆ H _5- ).
[0047]
[Example 1]
Induction of KDNase by KDN Derivatives Bacterial cells of sphingobacteria mOL12-4s were treated with 1% (w / v) casein hydrolyzate (casamino acids, manufactured by Gibco) and 1% (w / v) glucose (Glc). The cells were cultured at 25 ° C. in the M9 liquid medium containing. The growing cells (cells) were collected and washed twice with M9 liquid medium. Inoculate 1 × 10 ⁹ cells in 2.0 ml each of M9 liquid medium containing each of the additive substances shown in Table 1 at a concentration of 0.1% (w / v) and incubate at 25 ° C. for 12 hours, Intracellular KDNase activity was measured. For intracellular KDNase activity, cells collected by centrifuging the culture solution at 13,000 rpm (14,000 × g) for 10 minutes were suspended in 0.25 ml of 0.1 M Tris-acetate buffer (pH 6.0) containing 0.1 M NaCl. Ultrasonicated (20 watts, 30 seconds). The KDNase activity of the supernatant obtained after centrifugation at 13,000 rpm (14,000 × g) for 10 minutes was measured and calculated as a unit per 2 ml of culture solution. For KDNase activity, 1.4 nmol of KDNα2MeUmb as a substrate was added to 20 μl of the supernatant, reacted at 25 ° C. for 30 minutes, and after the reaction, mixed with 2.5 ml of 85 mM glycine carbonate buffer (pH 9.3). The fluorescence intensity was measured. The amount of enzyme that hydrolyzes 1 nmol of KDNα2MeUmb per minute was defined as 1 unit. The results are shown in Table 1.
[0048]
[Table 1]
Table 1 KDNase activity when various substances are added ────────────────────────────────
Additives KDNase activity (unit / 2 ml culture medium)
────────────────────────────────
KDNα2Me + Glc 7.3
KDNα2Et + Glc 11.0
KDNα2Bn + Glc 5.1
KDNα2pNP + Glc 9.0
KDNα2MeUmb + Glc 9.4
KDN-OS + Glc 2.1
KDN + Glc 0.3
Glc 0.7
KDNα2Bn 0.2
KDN-OS 4.5
KDN 0.1
────────────────────────────────
Note) The activity when KDNα2Me, KDNα2Et, KDNα2pNP and KDNα2MeUmb were added alone was 0.3 to 1.8, which was almost the same as KDNα2Bn.
[0049]
From Table 1, it is clear that the KDN derivative represented by the general formula (1) exhibits remarkable KDNase-inducing activity when glucose is present. In addition, glucose is added as a carbon source under general culture conditions because glucose exhibits the KDNase-inducing activity of the KDN derivative represented by the general formula (1) only in the presence of 0.1% (w / v). It can be seen that it is sufficient to add only the KDN derivative represented by the general formula (1) as a KDNase inducer under the condition where a level of glucose exists. Furthermore, it can be seen that under such conditions, the KDNase-inducing activity of the KDN derivative represented by the general formula (1) is significantly higher than that of KDN-OS.
[0050]
[Example 2]
Optimization of KDNase induction conditions When KDNα2MeUmb was used as an inducer, the conditions for inducing KphNase of sphingobacterium mOL12-4s were examined.
[0051]
(a) cultured at 25 ° C. in M9 liquid medium containing 1% (w / v) casein hydrolyzate (casamino acids, manufactured by Gibco) and 1% (w / v) glucose (Glc) Cells were seeded at various cell densities in M9 liquid medium containing 0.1% (w / v) KDNα2MeUmb and 0.1% (w / v) Glc and incubated at 25 ° C. for 12 hours, Example 1 Intracellular KDNase activity was measured by the same method. The results are shown in FIG. The optimal initial cell density for induction was 1.0 × 10 ⁹ cells / ml.
[0052]
(b) Cultivation at 25 ° C. in M9 liquid medium containing 1% (w / v) inducer concentration (casamino acids, manufactured by Gibco) and 1% (w / v) glucose (Glc) Cells were seeded in M9 liquid medium containing various concentrations of KDNα2MeUmb and 0.1% (w / v) Glc at a cell density of 1.0 × 10 ⁹ cells / ml, incubated at 25 ° C. for 12 hours, Intracellular KDNase activity was measured in the same manner as in Example 1. The results are shown in FIG. It was effective in inducing KDNase at a concentration of 0.01% (w / v).
(c) Cells cultured at 25 ° C. in M9 liquid medium containing 1% (w / v) induction time (casamino acids, manufactured by Gibco) and 1% (w / v) glucose (Glc) Is inoculated at a cell density of 1.0 × 10 ⁹ cells / ml in M9 liquid medium containing 0.1% (w / v) KDNα2MeUmb and 0.1% (w / v) Glc. And the intracellular KDNase activity was measured in the same manner as in Example 1. The results are shown in FIG. Effective KDNase induction was obtained in 6 to 24 hours.
[0053]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, the inducer of KDNase with a high KDNase induction effect and the manufacturing method of KDNase using the same are provided, and KDNase can be manufactured efficiently. The KDN derivative, which is an active ingredient of the inducer of the present invention, is easy to prepare and can be provided in a large amount at a low cost.
[Brief description of the drawings]
FIG. 1 shows a synthesis scheme of a KDN derivative.
FIG. 2 shows an example of KDNase induction conditions.

Claims (6)

A deaminoneuraminidase inducer for inducing deaminoneuraminidase produced by a bacterium belonging to the genus Sphingobacteria, which contains a deaminoneuraminic acid derivative represented by the following general formula (1) and glucose as active ingredients.

(R represents an alkyl group having 1 to 10 carbon atoms, an aralkyl group, or an aryl group.) The inducer according to claim 1 , wherein R is an alkyl group having 1 to 4 carbon atoms, a benzyl group, or a phenyl group or umbelliferyl group which may have a substituent. The inducer according to claim 2 , wherein R is an alkyl group having 1 to 2 carbon atoms, a nitrophenyl group, or a methylumbelliferyl group. In a medium containing an inducing agent according to any one of claims 1 to 3 and a bacterium belonging to Sphingomonas genus step of culturing in the production conditions of the deaminoneuraminidase, and, from the culture obtained by culturing A method for producing deaminoneuraminidase, comprising at least a step of collecting deaminoneuraminidase. The manufacturing method of Claim 4 including the following process at least.
(Step 1) The inducer according to any one of claims 1 to 3 is added to a culture containing a bacterium belonging to the genus Sphingobacterium.
(Step 2) The culture obtained in Step 1 is cultured under the deaminoneuraminidase production conditions.
(Step 3) Deaminoneuraminidase is collected from the culture obtained in Step 2. The production method according to claim 4 or 5 , wherein the bacterium belonging to the genus Sphingobacterium is sphingobacteria mOL12-4s (FERM BP-5116).

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